The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F. H., L. Kim 1988! TIBTECH 6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Until the last fifteen years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis var. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces .delta.-endotoxin crystals which are toxic to the larvae of a number of lepidopteran insects.
In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely B.t. var. israelensis and B.t. var. tenebrionis (a.k.a. M-7, a.k.a. B.t. var. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. 1989! "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255). See also Couch, T. L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76; and Beegle, C. C., (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104. Krieg et al. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter 1983! Z. ang. Ent. 96:500-508), describe Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and the beetle Agelastica alni.
Recently, new subspecies of B.t. have been identified, and genes responsible for active .delta.-endotoxin proteins have been isolated (Hofte, H., H. R. Whiteley 1989! Microbiological Reviews 52(2):242-255). Hofte and Whiteley classified B.t. crystal protein genes into 4 major classes. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). Prefontaine et al. (Prefontaine, G., P. Fast, P. C. K Lau, M. A. Hefford, Z. Hanna, R. Brosseau 1987! Appl. Environ. Microbiol. 53(12):2808-2814) describe probes useful in classifying lepidopteran-active genes. The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim 1992! Bio/Technology 10:271-275). B.t. crystalline toxins are generally recognized as being protoxins, requiring either particular physicochemical conditions (ie., pH, redox, ionic strength), or the action of certain proteases, or both, to generate an active toxin (Hofte and Whiteley, supra). In most cases, the insect supplies conditions for activation of the toxin; however, cases have been documented where pre-solubilization or pre-proteolysis have been necessary for optimum activity (Jacquet, J., R. Hutter, P. Luthy 1987! "Specificity of Bacillus thuringiensis delta-endotoxin," Appl. Environ. Microbiol. 53:500-504) or detection of activity (Lambert, B., H. R. Hofte, K. Annys, S. Jansens, P. Soetaert, M. Peferoen 1992! "Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae," Appl. Environ. MicrobioL 58:2536-2542).
The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H. E., H. R. Whiteley 1981! Proc. Natl. Acad. Sci. U.S.A. 78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of B.t. crystal proteins in E. coli. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis var. tenebrionis (a.k.a. B.t. san diego, a.k.a. M-7) which can be used to control coleopteran pests in various environments. U.S. Pat. No. 4,918,006 discloses Bacillus thuringiensis var. israelensis toxins which are active against dipteran pests and reports that a protein of about 27 kDa, and fragments thereof, are responsible for the dipteran activity. U.S. Pat. No. 4,849,217 discloses B.t. isolates which have activity against the alfalfa weevil. U.S. Pat. No. 5,151,363 and U.S. Pat. No. 4,948,734 disclose certain isolates of B.t. which have activity against nematodes. As a result of extensive research and investment of resources, other patents have issued for new B.t. isolates and new uses of B.t. isolates. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.
Hemiptera represent a major group of insects that have not, to date, been effectively controlled by B.t. .delta.-endotoxins. Numerous hemipteran pest species, most notably Lygus species, cause considerable plant damage and economic loss each year.
Hemiptera ranks among the most economically destructive orders. See Arnett, R. H. Jr. (1985! American Insects, Van Nostrand Reinhold Co., Inc., New York). Of all Hemiptera, the mirids (Hemiptera: Miridae; includes Lygus) are the most notorious crop pests. Feeding causes injury that weakens plants and is a mode of plant disease transmission. In addition to L. hesperus, other pest Lygus include: L. lineolaris (Beauv.), L. pratensis (L.), L. rugulipennis Popp., and the common green capsid (Lygus pabulinus (L.)). Members of this genus are found variously on cotton, potato, sugar beet, celery, beans, peach, apple, alfalfa, pear, plum, quince and a variety of nursery stock, ornamental plants and vegetable crops. Specific Mirid pests include: the potato capsid Calocoris norvegicus (Gmel.), a pest of potato and brassicas (e.g., cabbage, cauliflower, broccoli, kale, Brussels sprout, turnip); the stack bug (also carrot plant bug) Orthops campestris (L.), a pest of carrot, celery, parsnip, parsley and dill; the apple capsid Plesiocoris rugicollis (Fall.), a pest of apple, currants and gooseberry; the tomato bug Cyrtopeltis modestus (Distant), a pest of tomatoes; the suckfly Cyrtopeltis notatus (Distant), a pest of tobacco; the white marked fleahopper Spanagonicus albofasciatus (Reuter), injurious to grass, with damage particularly noticeable on golf greens; the honeylocust plant bug Diaphnocors chlorionis (Say), a pest of beets; the onion plant bug Labopidicola allii Knight, a pest of cultivated and wild onions; the cotton fleahopper Pseudatomoscelis seriatus (Reuter), found on cotton; the rapid plant bug Adelphocoris rapidus (Say), an occasional pest of cotton and legumes; the four-lined plant bug Poecilocapsus lineatus (Fabricius), often a pest of garden crops.
Other Hemipteran pests include:
Lygaeidae (seed bug family): chinch bug (Blissus leucopterus (Say)) is a pest of maize, sorghums, wheat, millets, rice, barley, rye and oats; false chinch bugs (Nysius spp., e.g., N. ericae, N. raphanus Howard) are pests of brassicas, beets and potato.
Pentatomidae (stink bug family): the brown stink bug, Euschistus servus (Say) is a pest of cotton. The green stink bug, Nezara viridula (L.) is a generalist pest of seedlings, especially vegetables and legumes; Eurygaster species, e.g. E. austiaca (Schr.) and E. integriceps (Put.) (wheat shield bug, Sunn pest, Senn bug) attack wheat and barley.
Coreidae (squash bug family): Anasa tristis (DeGreer) (squash bug) is a local pest of squash; A. armigera (Say) (homed squash bug) is an occasional pest of cultivated cucumber.
Pyrrhocoridae (red bug and cotton stainer family): the cotton stainer Dysdercus suturellus (Herrich-Schaeffer) is a pest of cotton.
Tingidae (lace bug family): Corythucha arcuata (Say) often is a pest of roses, maple, apple and chestnut. Other Corythucha species include the cotton lace bug, chrysanthemum lace bug, elm lace bug and hawthorn lace bug.
Belostomatidae (giant water bug family): members of this family are known to attack and suck the blood of fishes and therefore can be a pest in fish hatcheries.
Members of the Reduviidae and Cimicidae (including kissing bug and bed bugs, respectively) bite mammals and transmit diseases contractible by humans.
The digestive system of hemipterans is unusual among the insects in several ways: certain hydrolytic digestive enzymes are absent such as trypsin; the midgut lacks a peritrophic membrane, and there is no crop. These features reflect the liquid diet and sucking mode of feeding, subject to evolutionary constraints. Because of the differences in diet, feeding mode, and digestive physiology and biochemistry, one would not necessarily expect that proteins having insecticidal activity against leaf-chewing insects would also have activity against fluid-feeding Hemiptera.
Bacillus thuringiensis PS201T6, NRRL B-18750, deposited on Jan. 9, 1991, or a .delta.-endotoxin therefrom, was previously found to have activity against certain pests. For example, see U.S. Pat. Nos. 5,273,746; 5,298,245; and 5,302,387, which disclose the use of B.t. PS201T6. B.t. isolates PS123D1 has been disclosed in EP 0 409 438. B.t. isolate PS71M3 has been disclosed in EP 0 626 809 and in U.S. Pat. No. 5,273,746. The above patents do not disclose or suggest the use of any B.t. isolate to control hemipteran insect pests.
Practice of the present invention provides an alternative to control of hemipteran pests with chemical pesticides, thereby allowing more environmentally-friendly insect management and providing a tool for management of insecticide resistance.